The basis for haplotype complexity in VCBPs, an immune-type receptor in amphioxus

Innate immune gene repertoires are restricted primarily to germline variation. Adaptive immunity, by comparison, relies on somatic variation of germline-encoded genes to generate extraordinarily large numbers of non-heritable antigen recognition motifs. Invertebrates lack the key features of vertebrate adaptive immunity, but have evolved a variety of alternative mechanisms to successfully protect the integrity of “self”; in many cases, these appear to be taxon-specific innovations. In the protochordate Branchiostoma floridae (amphioxus), the variable region-containing chitin-binding proteins (VCBPs) constitute a multigene family (comprised of VCBPs 1–5), which possesses features that are consistent with innate immune-type function. A large number of VCBP alleles and haplotypes are shown to exhibit levels of polymorphism exceeding the elevated overall levels determined for the whole amphioxus genome (JGI). VCBP genes of the 2 and 5 types are distinguished further by a highly polymorphic segment (exon 2) in the N-terminal immunoglobulin domain, defined previously as a “hypervariable region” or a “hotspot.” Genomic deoxyribonucleic acid (DNA) and complementary DNA (cDNA) sequences from large numbers of animals representing different populations reveal further significant differences in sequence complexity within and across VCBP2/5 haplotypes that arise through overlapping mechanisms of genetic exchange, gene copy number variation as well as mutation and give rise to distinct allelic lineages. The collective observations suggest that mechanisms were in place at the time of divergence of the cephalochordates that could selectively hyperdiversify immune-type receptors within a multigene family.     

Expanding our understanding of immunoglobulin, T-cell antigen receptor, and novel immune-type receptor genes: a subset of the immunoglobulin gene superfamily

 The immunoglobulin superfamily (IgSF) is an extensively diversified multigene family whose members share a common structural feature, the Ig fold. Members of the Ig/T-cell antigen receptor (TCR) subset of the IgSF mediate antigen-specific recognition in adaptive immune responses. Antigen-binding receptors belonging to this subset are present in all species of jawed vertebrates. To explore whether there are additional structurally related but otherwise distinct members of this subset, we have developed a technique termed the short-primer polymerase chain reaction (PCR) that targets structurally conserved short motifs in the Ig fold. Large-scale sequencing efforts and recent advances in information biotechnology, including "electronic PCR," provide additional computational means to implement similarly directed searches within databases. The use of these approaches has led to the discoveries of Ig/TCR homologues in a variety of phylogenetically diverse organisms, a diversified family of novel immune-type receptor genes, as well as a novel human IgSF member. The potential of random sequencing efforts and virtual screening of databases is described in the context of two novel genes in bony fish. The various methodologies that are discussed and the examples shown provide means for further investigating, and/or elucidating novel, IgSF receptors as well as components of pathways that are involved in immune responses in both traditional and nontraditional model systems.     

Novel Immune-type Receptor Genes and the Origins of Adaptive and Innate Immune Recognition

The prototypic forms of teleost novel immune-type receptors(NITRs) consist of a variable (V) region, a unique V-like C2(V/C2) domain, a transmembrane region and a cytoplasmic tailcontaining immunoreceptor tyrosine-based inhibition motifs (ITIMs).NITRs encode diversified V regions in large multigene familiesbut do not undergo somatic rearrangement. Studies in four differentbony fish model systems have identified a number of differentorganizational forms of NITRs. Specifically, NITR genes encodeN-terminal ectodomains of the V-type but otherwise vary in the:total number of extracellular immunoglobulin domains, numberand location of joining (J) region-like motifs, presence oftransmembrane regions, presence of charged residues within transmembraneregions, presence of cytoplasmic tails, and/or distributionof ITIM(s) within the cytoplasmic tails. V region-containingNITRs constitute a far more complex family than recognized originallyand currently include individual members that potentially functionthrough inhibitory as well as activating mechanisms. The genomicorganization of the NITR gene cluster as well as the structuraldiversity and overall architecture of the NITR proteins is reminiscentof genes encoded at the mammalian leukocyte receptor cluster(LRC); however, there presently is no functional evidence tosupport an orthologous relationship between NITR and LRC geneproducts. Comparisons of the predicted structures of the NITRshave identified several short regions of sequence identity anda novel cloning strategy has been devised that selects for secretoryand transmembrane proteins that encode these short motifs. Usingthis approach, related genes termed immune-type receptors (ITRs)have been identified in cartilaginous fish. Taken together,these studies indicate that leukocyte regulatory receptors,including those that mediate natural killer function, mighthave emerged early in vertebrate evolution and that the NITR/ITRgenes represent a new and potentially highly significant linkbetween innate and adaptive immune responses.     

Evidence for a transposition event in a second NITR gene cluster in zebrafish

Novel immune-type receptors (NITRs) are immunoglobulin-variable (V) domain-containing cell surface proteins that possess characteristic activating/inhibitory signaling motifs and are expressed in hematopoietic cells. NITRs are encoded by multigene families and have been identified in bony fish species. A single gene cluster, which encodes 36 NITRs that can be classified into 12 families, has been mapped to zebrafish chromosome 7. We report herein the presence of a second NITR gene cluster on zebrafish chromosome 14, which is comprised of three genes (nitr13, nitr14a, and nitr14b) representing two additional NITR gene families. Phylogenetic analyses indicate that the V domains encoded by the nitr13 and nitr14 genes are more similar to each other than any other zebrafish NITR suggesting that these genes arose from a tandem gene duplication event. Similar analyses comparing zebrafish Nitr13 and Nitr14 to NITRs from other fish species indicate that the nitr13 and nitr14 genes are phylogenetically related to the catfish IpNITR13 and IpNITR15 genes. Sequence features of the chromosomal region encoding nitr13 suggest that this gene arose via retrotransposition.     

Developmental and tissue-specific expression of NITRs

Novel immune-type receptors (NITRs) are encoded by large multi-gene families and share structural and signaling similarities to mammalian natural killer receptors (NKRs). NITRs have been identified in multiple bony fish species, including zebrafish, and may be restricted to this large taxonomic group. Thirty-nine NITR genes that can be classified into 14 families are encoded on zebrafish chromosomes 7 and 14. Herein, we demonstrate the expression of multiple NITR genes in the zebrafish ovary and during embryogenesis. All 14 families of zebrafish NITRs are expressed in hematopoietic kidney, spleen and intestine as are immunoglobulin and T cell antigen receptors. Furthermore, all 14 families of NITRs are shown to be expressed in the lymphocyte lineage, but not in the myeloid lineage, consistent with the hypothesis that NITRs function as NKRs. Sequence analyses of NITR amplicons identify known alleles and reveal additional alleles within the nitr1, nitr2, nitr3, and nitr5 families, reflecting the recent evolution of this gene family.     

Cloning novel immune-type inhibitory receptors from the rainbow trout, <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>

Novel immune-type receptor ( NITR) genes that encode two extracellular immunoglobulin domains and cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been described previously in three lineages of bony fish. In the current study, four ITIM-containing NITR cDNAs are identified in the rainbow trout ( Oncorhynchus mykiss), and their expression patterns and genomic complexity are characterized. The ITIM-containing NITR2 gene maps 1.3 cM from an ITIM-containing C-type lectin receptor ( TCL-2) on linkage group XXI. A comprehensive, phylogenetic analysis of NITRs from rainbow trout and three other major lineages of bony fish defines conserved families of NITRs and suggests an ancient lineage of distinct groups of genes. Several probable scenarios that explain the origins of variant forms of NITRs are described.     

Novel immunoglobulin-like transcripts in teleost fish encode polymorphic receptors with cytoplasmic ITAM or ITIM and a new structural Ig domain similar to the natural cytotoxicity receptor NKp44

Members of the immunoglobulin superfamily (IgSF) include a group of innate immune receptors located in the leukocyte receptor complex (LRC) and other small clusters such as the TREM/NKp44 cluster. These receptors are characterised by the presence of immunoglobulin domains, a stalk, a transmembrane domain, and a cytoplasmic region containing either an immunoreceptor tyrosine-based inhibitory motif (ITIM) or are linked to an adapter molecule with an activation motif (ITAM) for downstream signalling. We have isolated two carp cDNA sequences encoding receptors in which the extracellular Ig domain structurally resembles the novel V-type Ig domain of NKp44. This is supported by a homology model. The cytoplasmic regions contain either an ITAM (Cyca-NILT1) or ITIMs (Cyca-NILT2). The tissue expression of these receptors is nearly identical, with the highest expression in the immunological organs. Peripheral blood leucocytes showed no detectable expression, but upon in vitro culture expressed NILT1, the activating receptor, and not the inhibitory NILT2 receptor. Southern blot analysis indicated that the NILT1 and NILT2 sequences belong to a multigene family. Analysis of the NILT Ig domain-encoding sequences amplified from both genomic DNA and cDNA revealed extensive haplotypic and allelic polymorphism. Database mining of the zebrafish genome identified several homologs on Chromosome 1, which also contains a cluster of class I major histocompatibility genes. This constellation is reminiscent of the TREM/NKp44 gene cluster and the HLA complex located on human Chromosome 6. The carp NILT genes form a unique cluster of innate immune receptors, which are highly polymorphic, and characterised by a new Ig structural subfamily and are distinct from the novel immune-type receptors (Nitrs) found in other fish species.     

The amphioxus genome provides unique insight into the evolution of immunity

Immune systems evolve as essential strategies to maintain homeostasis with the environment, prevent microbial assault and recycle damaged host tissues. The immune system is composed of two components, innate and adaptive immunity. The former is common to all animals while the latter consists of a vertebrate-specific system that relies on somatically derived lymphocytes and is associated with near limitless genetic diversity as well as long-term memory. Deuterostome invertebrates provide a view of immune repertoires in phyla that immediately predate the origins of vertebrates. Genomic studies in amphioxus, a cephalochordate, have revealed homologs of genes encoding most innate immune receptors found in vertebrates; however, many of the gene families have undergone dramatic expansions, greatly increasing the innate immune repertoire. In addition, domain-swapping accounts for the innovation of new predicted pathways of receptor function. In both amphioxus and Ciona, a urochordate, the VCBPs (variable region containing chitin-binding proteins), which consist of immunoglobulin V (variable) and chitin binding domains, mediate recognition through the V domains. The V domains of VCBPs in amphioxus exhibit high levels of allelic complexity that presumably relate to functional specificity. Various features of the amphioxus immune repertoire reflect novel selective pressures, which likely have resulted in innovative strategies. Functional genomic studies underscore the value of amphioxus as a model for studying innate immunity and may help reveal how unique relationships between innate immune receptors and both pathogens and symbionts factored in the evolution of adaptive immune systems.     

The phylogenetic origins of natural killer receptors and recognition: relationships,possibilities, and realities

Natural killer (NK) cells affect a form of innate immunity that recognizes and eliminates cells that are infected with certain viruses or have undergone malignant transformation. In mammals, this recognition can be mediated through immunoglobulin- (Ig) and/or lectin-type NK receptors (NKRs). NKR genes in mammals range from minimally polymorphic single-copy genes to complex multigene families that exhibit high levels of haplotypic complexity and exhibit significant interspecific variation. Certain single-copy NKR genes that are present in one mammal are present as expanded multigene families in other mammals. These observations highlight NKRs as one of the most rapidly evolving eukaryotic gene families and likely reflect the influence of pathogens, especially viruses, on their evolution. Although well characterized in human and mice, cytotoxic cells that are functionally similar to NK cells have been identified in species ranging from birds to reptiles, amphibians and fish. Although numerous receptors have been identified in non-mammalian vertebrates that share structural relationships with mammalian NKRs, functionally defining these lower vertebrate molecules as NKRs is confounded by methodological and interpretive complexities. Nevertheless, several lines of evidence suggest that NK-type function or its equivalent has sustained a long evolutionary history throughout vertebrate species.     

Identification and characterisation of a novel immune-type receptor (NITR) gene cluster in the European sea bass, Dicentrarchus labrax, reveals recurrent gene expansion and diversification by positive selection

In the last decade, a new gene family encoding non-rearranging receptors, called novel immune-type receptors (NITRs), has been discovered in teleost fish. NITRs belong to the immunoglobulin superfamily and represent an extraordinarily divergent and rapidly evolving gene complex. Genomic analysis of a region spanning 270 kb led to the discovery of a NITR gene cluster in the European sea bass (Dicentrarchus labrax). In total, 27 NITR genes and three putative pseudogenes, organised in a tandemly arrayed cluster, were identified. Sea bass NITR genes maintain the three major genomic organisations that appear to be essentially conserved among fish species along with new features presumably involving processes of intron loss, exon deletion and acquisition of new exons. Comparative and evolutionary analyses suggest that these receptors have evolved following a “birth-and-death” model of gene evolution in which duplication events together with lineage-specific gain and loss of individual members contributed to the rapid diversification of individual gene families. In this study, we demonstrate that species-specific gene expansions provide the raw material for diversifying, positive Darwinian selection favouring the evolution of a highly diverse array of molecules.     

A novel soluble immune-type receptor (SITR) in teleost fish: carp SITR is involved in the nitric oxide-mediated response to a protozoan parasite

Background

The innate immune system relies upon a wide range of germ-line encoded receptors including a large number of immunoglobulin superfamily (IgSF) receptors. Different Ig-like immune receptor families have been reported in mammals, birds, amphibians and fish. Most innate immune receptors of the IgSF are type I transmembrane proteins containing one or more extracellular Ig-like domains and their regulation of effector functions is mediated intracellularly by distinct stimulatory or inhibitory pathways.

Methodology/Principal Findings

Carp SITR was found in a substracted cDNA repertoire from carp macrophages, enriched for genes up-regulated in response to the protozoan parasite Trypanoplasma borreli. Carp SITR is a type I protein with two extracellular Ig domains in a unique organisation of a N-proximal V/C2 (or I-) type and a C-proximal V-type Ig domain, devoid of a transmembrane domain or any intracytoplasmic signalling motif. The carp SITR C-proximal V-type Ig domain, in particular, has a close sequence similarity and conserved structural characteristics to the mammalian CD300 molecules. By generating an anti-SITR antibody we could show that SITR protein expression was restricted to cells of the myeloid lineage. Carp SITR is abundantly expressed in macrophages and is secreted upon in vitro stimulation with the protozoan parasite T. borreli. Secretion of SITR protein during in vivo T. borreli infection suggests a role for this IgSF receptor in the host response to this protozoan parasite. Overexpression of carp SITR in mouse macrophages and knock-down of SITR protein expression in carp macrophages, using morpholino antisense technology, provided evidence for the involvement of carp SITR in the parasite-induced NO production.

Conclusion/Significance

We report the structural and functional characterization of a novel soluble immune-type receptor (SITR) in a teleost fish and propose a role for carp SITR in the NO-mediated response to a protozoan parasite.     

Genomic and functional characterization of the diverse immunoglobulin domain-containing protein (DICP) family

A heretofore-unrecognized multigene family encoding diverse immunoglobulin (Ig) domain-containing proteins (DICPs) was identified in the zebrafish genome. Twenty-nine distinct loci mapping to three chromosomal regions encode receptor-type structures possessing two classes of Ig ectodomains (D1 and D2). The sequence and number of Ig domains, transmembrane regions and signaling motifs vary between DICPs. Interindividual polymorphism and alternative RNA processing contribute to DICP diversity. Molecular models indicate that most D1 domains are of the variable (V) type; D2 domains are Ig-like. Sequence differences between D1 domains are concentrated in hypervariable regions on the front sheet strands of the Ig fold. Recombinant DICP Ig domains bind lipids, a property shared by mammalian CD300 and TREM family members. These findings suggest that novel multigene families encoding diversified immune receptors have arisen in different vertebrate lineages and affect parallel patterns of ligand recognition that potentially impact species-specific advantages.     

Adaptive significance of the limited gene expression of the immune system

Evolutionary development of immunity multigene systems seems to involve host-parasite molecular coevolution, as evidenced by comparison of metasoans and unicellular organisms in fixation rates of neutral and adaptive point mutations per one pair of corresponding genes. The estimations we attempted revealed that organisms with maximal restriction of Ig-like gene repertoire expression by an individual immune cell are more adaptive in formation of competent antigen-induced immune response, provided that the magnitude of potential repertoire of functional Ig-like receptors exceeds lymphoid cell population size. In contrast, when phage and bacteria populations are in coevolution, there is no need to limit the repertoire of expressive genes encoding bacteria surface receptor proteins which are recognized by a phage adsorption system. Other hypotheses of evolutionary premises and stimulus of immune genes expression limitation (allelic, isotypic etc.) are critically discussed.     

A novel family of diversified immunoregulatory receptors in teleosts is homologous to both mammalian Fc receptors and molecules encoded within the leukocyte receptor complex

Three novel and closely related leukocyte immune-type receptors (IpLITR) have been identified in channel catfish (Ictalurus punctatus). These receptors belong to a large polymorphic and polygenic subset of the Ig superfamily with members located on at least three independently segregating loci. Like mammalian and avian innate immune regulatory receptors, IpLITRs have both putative inhibitory and stimulatory forms, with multiple types coexpressed in various lymphoid tissues and clonal leukocyte cell lines. IpLITRs have an unusual and novel relationship to mammalian and avian innate immune receptors: the membrane distal Ig domains of an individual IpLITR are related to fragment crystallizable receptors (FcRs) and FcR-like proteins, whereas the membrane proximal Ig domains are related to several leukocyte receptor complex encoded receptors. This unique composition of Ig domains within individual receptors supports the hypothesis that functionally and genomically distinct immune receptor families found in tetrapods may have evolved from such ancestral genes by duplication and recombination events. Furthermore, the discovery of a large heterogeneous family of immunoregulatory receptors in teleosts, reminiscent of amphibian, avian, and mammalian Ig-like receptors, suggests that complex innate immune receptor networks have been conserved during vertebrate evolution.Electronic supplementary material Supplementary material is available for this article at and is accessible for authorized users. GenBank Submissions: The sequences presented in this article have been submitted to GenBank under the following accession numbers: AAW82352, IpLITR1; AAW82353, IpLITR2; AAW82354, IpLITR3.     

Hagfish leukocytes express a paired receptor family with a variable domain resembling those of antigen receptors

Jawed vertebrates are equipped with TCR and BCR with the capacity to rearrange their V domains. By contrast, jawless vertebrates, represented by hagfish and lampreys, apparently lack such receptors. We describe in this study a family of hagfish genes carrying a single V-type domain resembling those of TCR/BCR. This multigene family, which we call agnathan paired receptors resembling Ag receptors (APAR), is expressed in leukocytes and predicted to encode a group of membrane glycoproteins with organizations characteristic of paired Ig-like receptors, consisting of activating and inhibitory forms. APAR has a J region in its V-type domain, and its V and J regions are encoded in a single exon. Thus, APAR is a member of the emerging families of diversified, innate immune-type receptors with TCR/BCR-like V-type domains and has many of the features expected for a primordial TCR/BCR-like receptor. The extracellular domain of APAR may be descended from a V-type domain postulated to have acquired recombination signal sequences in a jawed vertebrate lineage.     

Members of the Ikaros gene family are present in early representative vertebrates

Members of the Ikaros multigene family of zinc finger proteins are expressed in a tissue-specific manner and most are critical determinants in the development of both the B and T lymphocytes as well as NK and dendritic APC lineages. A PCR amplification strategy that is based on regions of shared sequence identity in Ikaros multigene family members found in mammals and several other vertebrates has led to the recovery of cDNAs that represent the orthologues of Ikaros, Aiolos, Helios, and Eos in Raja eglanteria (clearnose skate), a cartilaginous fish that is representative of an early divergence event in the phylogenetic diversification of the vertebrates. The tissue-specific patterns of expression for at least two of the four Ikaros family members in skate resemble the patterns observed in mammals, i.e., in hematopoietic tissues. Prominent expression of Ikaros in skate also is found in the lymphoid Leydig organ and epigonal tissues, which are unique to cartilaginous fish. An Ikaros-related gene has been identified in Petromyzon marinus (sea lamprey), a jawless vertebrate species, in which neither Ig nor TCRs have been identified. In addition to establishing a high degree of evolutionary conservation of the Ikaros multigene family from cartilaginous fish through mammals, these studies define a possible link between factors that regulate the differentiation of immune-type cells in the jawed vertebrates and related factors of unknown function in jawless vertebrates.     

Characterization of additional novel immune type receptors in channel catfish, <Emphasis Type="Italic">Ictalurus punctatus</Emphasis>

Mining of channel catfish (Ictalurus punctatus) expressed sequence tag databases identified seven new novel immune type receptors (IpNITRs). These differed in sequence, but not structure, from previously described IpNITR1-11. IpNITR12a, 12b, 13, and 14 encode proteins containing a single variable (V)-like immunoglobulin (Ig) domain. IpNITR12a and 13 encode a transmembrane (TM) region and cytoplasmic tail (CYT) containing immunoreceptor tyrosine inhibition motifs (ITIMs). IpNITR14 contains a TM and short CYT devoid of signaling motifs and is similar in structure to IpNITR7. IpNITR12b lacks a TM and may represent an IpNITR12a splice variant. In contrast, IpNITR15a, 15b, and 16 encode two Ig domains (V-like domain 1 and V/C2-like domain 2). IpNITR15a and 15b contain TM and CYT with ITIMs. IpNITR16 appears to be a secreted form. The first V-like domains of IpNITR12-16 (except a/b pairs) share 17–32% amino acid identity with each other and with V domains of IpNITR1-11. They therefore represent five additional IpNITR V families (defined as possessing 70% or more amino acid identity). The V/C2 domains of IpNITR15a, 15b and 16 have 94–98% amino acid identity, but share 37–50% amino acid identity with corresponding V/C2 domains found in IpNITR1-4. Phylogenetic analyses indicate IpNITR12-16 are more closely related to other teleost NITRs than to IpNITR1-11. Gene mapping indicates that IpNITRs are linked, and members of the ten known IpNITR families are interspersed. IpNITR12-16 are differentially expressed in various catfish immune-type cells and preferentially up regulated in peripheral blood leukocytes by allogeneic stimulation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. GenBank Submissions: The sequences presented in this article have been submitted to GenBank under the following accession numbers: IpNITR12a, EF490801; IpNITR12b, EF490802; IpNITR13, EF490803; IpNITR14, EF490804; IpNITR15a, EF490805; IpNITR15b, EF490806; and IpNITR16, EF490807.     

Did the molecules of adaptive immunity evolve from the innate immune system?

The antigen receptors on cells of innate immune systems recognizebroadly expressed markers on non-host cells while the receptorson lymphocytes of the adaptive immune system display a higherlevel of specificity. Adaptive immunity, with its exquisitespecificity and immunological memory, has only been found inthe jawed vertebrates, which also display innate immunity. Jawlessfishes and invertebrates only have innate immunity. In the adaptiveimmune response, T and B-lymphocytes detect foreign agents orantigens using T cell receptors (TCR) or immunoglobulins (Ig),respectively. While Ig can bind free intact antigens, TCR onlybinds processed antigenic fragments that are presented on moleculesencoded in the major histocompatibility complex (MHC). MHC moleculesdisplay variation through allelic polymorphism. A diverse repertoireof Ig and TCR molecules is generated by gene rearrangement andjunctional diversity, processes carried out by the recombinaseactivating gene (RAG) products and terminal deoxynucleotidyltransferase (TdT). Thus, the molecules that define adaptiveimmunity are TCR, Ig, MHC molecules, RAG products and TdT. Nodirect predecessors of these molecules have been found in thejawless fishes or invertebrates. In contrast, the complementcascade can be activated by either adaptive or innate immunesystems and contains examples of molecules that gradually evolvedfrom non-immune functions to being part of the innate and thenadaptive immune system. In this paper we examine the moleculesof the adaptive immune system and speculate on the existenceof direct predecessors that were part of innate immunity.